PROJECT SUMMARY Dopamine, norepinephrine, and serotonin are major modulatory neurotransmitters that are implicated in a wide variety of psychiatric and neurological disorders, including addiction. Our available methods to quantify the dynamics of these neurotransmitters in extracellular space are not as fast, sensitive, direct or as clean as we would like. Here we propose to leverage recent progress in nanoscience to solve this problem by moving new near infrared (nIR) nanosensor technology from the nanoscientists' bench into the neurobiologists' rig. For ease of calibration and strong relevance to addiction, we will start with a dopamine nanosensor. We will integrate new nIR nanosensor tools with existing imaging and recording methods in brain slices and intact mice to enable greater understanding of the biology of modulatory neurotransmission. In Specific Aim 1, we plan to develop and calibrate the use of dopamine sensitive nanosensors for ex vivo detection of evoked dopamine release in striatal and frontal cortex brain slices. In Specific Aim 2 we will test the feasibility of using these sensors over long time scales in vivo. In Specific Aim 3, we will image nanosensor response to evoked dopamine release in vivo in intact and potentially awake behaving mice. Our goal is to set the stage to optically monitor dopamine and other neurotransmitter levels in vivo, in response to cues and rewards in conditions which induce reward prediction error in mouse models of health and disease. Development and dissemination of the dopamine nanosensor alone will greatly inform our understanding of substances with abuse potential and the effects of a broad variety of pharmacological agents. Experimental data collected using the dopamine nanosensor will then be applied to facilitate development of the norepinephrine and serotonin sensors. Our ultimate goal is to develop methods for measuring dopamine, norepinephrine, and serotonin in the cortex in vivo simultaneously. New infrared nanosensors have the potential to greatly advance our understanding of the brain, granting us new eyes to see modulatory neurotransmission in real time.